Hepatocyte growth factor (HGF/SF) is a polypeptide identified and purified by Nakamura, T., et al., Biochem. Biophys. Res. Commun. 22 (1984) 1450-1459. It was further found that hepatocyte growth factor is identical to scatter factor (SF), Weidner, K. M., et al., Proc. Natl. Acad. Sci. USA 88 (1991) 7001-7005. HGF is a glycoprotein with a molecular weight of about 100 kDa involved in the development of a number of cellular phenotypes including proliferation, mitogenesis, formation of branching tubules and, in the case of tumor cells, invasion and metastasis. For a status review, see Stuart, K. A., et al., International Journal of Experimental Pathology 81 (2000) 17-30. Both rat HGF and human HGF have been sequenced and cloned (Miyazawa, K. et al., Biochem. Biophys. Res. Comm. 163 (1989) 967-973; Nakamura, T., et al., Nature 342 (1989) 440-443; Seki, T., et al., Biochem. and Biophys. Res. Comm. 172 (1990) 321-327; Tashiro, K., et al., Proc. Natl. Acad. Sci. USA 87 (1990) 3200-3204; Okajima, A., et al., Eur. J. Biochem. 193 (1990) 375-381). The pharmacokinetics and pharmacological effects of an HGF lacking the first five N-terminal amino acids (dHGF) were investigated by Uematsu, Y., et al., J. Pharm. Sciences 88 (1999) 131-135. It was found that the serum concentration of dHGF decreased rapidly and therefore infusion would be preferred against bolus injection as administration route.
U.S. Pat. No. 5,977,310 describes PEG-modified HGF. Such PEG-modified HGF has a prolonged clearance in vivo and has the same physiological activity as HGF. However, according to U.S. Pat. No. 5,977,310, it is only possible to prolong the half life of HGF from 59.2 minutes to 76.7 minutes or 95.6 minutes, respectively (see Example 5 of U.S. Pat. No. 5,977,310). It is further suggested in this patent that the molar amount of the PEG reagent may be selected from the range of from 5 to 100 times of the molar weight of HGF. In the case of modifying an amino group of lysine or the N-terminus of protein, a preferred molar range of the PEG reagent is of from 10 to 25 times of the molar weight of HGF. The molecular weight of the attached PEG chain was about 10 kDa. Methods for the synthesis of conjugates consisting of PEG and polypeptides such as HGF are also described in WO 94/13322. These conjugates are linked together at predefined positions as random conjugation leads, according to the authors, to the introduction of polymeric moieties into domains of the molecule that mediate the therapeutically or diagnostically desirable activities. Consequently, the molecules may acquire a prolonged half-life in vivo and, in the case of heterologous proteins, reduced immunogenicity, but at the expense of a significant or complete loss of the desired biological activities (see, e.g., Kitamura, K., et al., Cancer Res. 51 (1991) 4310-4315 and Maiti, P. K., et al., Int. J. Cancer Suppl. 3 (1988) 17-22). PEGylated IFN-α shows, for example, only 7% of the potency compared to non-PEGylated IFN-α (Bailon, P., Bioconjugate Chem. 12 (2001) 195-202).
It was further found that an HGF/SF fragment, termed NK4, consisting of the N-terminal hairpin domain and the four kringle domains of HGF/SF, has pharmacological properties that are completely different from those of HGF/SF, and is an antagonist to the influence of HGF/SF on the motility and the invasion of colon cancer cells, and is, in addition, an angiogenesis inhibitor that suppresses tumor growth and metastasis (WO 93/23541; Parr, C., et al., Int. J. Cancer 85 (2000) 563-570; Kuba, K., et al., Cancer Res. 60 (2000) 6737-6743; Date, K., et al., FEBS Letters 420 (1997) 1-6; Date, K., et al., Oncogene 17 (1989) 3045-3054; Tomioka, D., et al., Cancer Res. 61 (2001) 7518-7524).
As emerges from Kuba, K., et al., Cancer Res. 60 (2000) 6737-6743, in animal experiments, for detecting an effect of NK4 on lung metastases, NK4 had to be infused continuously over a period of two weeks.
It is known that the attachment of polymers to certain polypeptides may increase the serum half life of such polypeptides. This was found, for example, for PEGylated Interleukin-6 (EP 0 442 724) or Interleukin-2 (WO 90/07938) and erythropoietin (WO 01/02017). However, the attachment of polyethylene glycol and other polymers does not necessarily lead to prolongation of their serum half lives. It is known, for example, that the conjugation of different polyethylene glycols to Interleukin-8, G-CSF and other interleukins results in the production of molecules with impaired properties (Mehvar, R., J. Pharm. Pharm. Sci. 3 (1) (2000) 125-136). Thus, the outcome of a PEGylation of a polypeptide is highly unpredictable. Gaertner, H. F., and Offord, R. E., Bioconjugate Chem. 7 (1996) 38-44 describes the site-specific attachment of PEG to the amino terminus of proteins. Gaertner et al. state (as already mentioned in WO 94/13222, see above) that PEGylation presents a big problem if the attachment sites cannot be precisely controlled, as this might have important implications for protein stability and function.
Francis, G. E., et al., Int. J. Hematol. 68 (1998) 1-18 present an overview of PEGylation of cytokines and other therapeutic proteins. Francis et al. state that with the majority of methods of PEGylation, substantial reduction of bioactivity has been reported (typically, 20-95%). According to Francis et al., PEGylation of proteins is always based on trial and error and virtually all parameters of such a PEGylation can have a surprising and very profound effect on the functionality of the product. Tsutsumi, Y., et al., Thromb. Haemost. 77 (1997) 168-173 describes the PEGylation of Interleukin-6. According to Tsutsumi et al., about 54% of the lysine amino groups of IL-6 were coupled with PEG with a molecular weight of 5 kDa per PEG group. Tsutsumi et al., in Proc. Natl. Acad. Sci. USA 97 (2000) 8548-8553, describe the chemical modification of an immunotoxin by PEG. As random PEGylation was accompanied by a significant loss of specific cytotoxic activity, Tsutsumi performs a site-specific PEGylation by using an immunotoxin mutant with one or two additional cysteins which are used for PEG coupling. Heinzerling, L., et al., Dermatol. 201 (2000) 154-157 describes the coupling of PEG to Interferon-α with a molecular weight of 5 kDa. Tsutsumi, Y., et al., in J. Pharmacol. Exp. Ther. 278 (1996) 1006-1011, describe the PEG modification of TNF-α, whereby the molecular weight of the PEG groups used is again 5 kDa. As the PEGylated TNF-α applied has a molecular weight of at least 84 kDa (by a molecular weight of 17 kDa of TNF-α) there are at least 13 5-kDa PEG groups attached to TNF-α.
PEGylation of proteins and its pharmacological effects are also reviewed by Reddy, K. R., Ann. Pharmacotherapy 34 (2000) 915-923. Again it is stated that PEGylation of therapeutic proteins must be carefully evaluated. Each protein is, according to Reddy et al., different, requires different optimization chemistry and therefore the influence of PEGylation cannot be predicted.